Hox genes comprise an evolutionarily conserved family of transcription factors first described for their function in segmental body patterning during development. These genes have since been demonstrated to be important regulators of hematopoiesis and tissue homeostasis, and their misregulation is strongly linked to cancer. Like many transcription factors, Hox proteins can act as transcriptional activators or repressors depending on the target gene and cellular context, but how the sign of transcriptional regulation is determined remains unclear. A detailed characterization of the factors that work with Hox proteins to control gene expression has been lacking due to limitations in conventional genetic and biochemical methods. The Mann laboratory recently developed a novel technique called cell- and gene-specific chromatin immunoprecipitation (cgChIP) to isolate specific fragments of chromatin from specific subsets of cells in Drosophila. This technique will be extended to isolate Hox protein complexes associated with a single regulatory element in a single cell type. Proteomic profiling of single cis-regulatory elements targeted by individual Hox proteins is essential for understanding how Hox activity is defined in a context-dependent manner. Specific aims: The goal of this proposal is to test the hypothesis that 1) novel co-activators or co-repressors interact with Hox proteins to determine the sign of transcriptional activity, and that 2) the recruitment of these components is dependent on the Hox molecular architecture required for DNA target selection. To test this hypothesis, initial studies will focus on the mechanism by which the Drosophila Hox protein sex combs reduced (Scr) activates fkh transcription by the enhancer element fkh250 through the following specific aims: (1) Optimize the biochemical isolation of fkh250-activating Scr complexes from embryos by cell- and gene-specific chromatin immunoprecipitation (cgChIP). (2) Identify novel components of the fkh250-activating Scr complex and test Scr motifs required for binding. (3) Determine whether Scr-bound proteins are necessary for fkh250 activation in vivo. Study design: The cgChIP method will be used to purify the Scr complex specifically bound to fkh250 from Drosophila embryos. To identify components of this Scr complex, bound proteins will be analyzed by mass-spectrometry. To gain insight into how these proteins are recruited specifically to this locus, motifs in Scr that are required for their interaction will be mapped. Additional experiments will test whether these binding proteins function as transcriptional co- activators by analyzing mutant embryos for their ability to activate fkh250. PUBLIC HEALTH RELEVANCE: Misregulation of Hox transcription factors is linked to a number of cancers, including acute myeloid leukemia (AML) and breast cancer. However, targeting Hox genes for cancer therapy is complicated by tissue-specific differences in their activities and downstream genes, potentially causing unwanted side effects. Thus, the identification and characterization of Hox cofactors that regulate Hox activity in a cell- and gene-specific manner is critical to developing specific anti-cancer therapies.